We can get a better understanding of the functional laws of all the various systems of the organism by studying the biological mechanisms implicated in their interaction and mutual impact. The significance of these factors can be well seen in our experience of conjoint research into the hemopoietic and immune systems.

Fundamentals of the theory of immunological regulation of hemopoiesis (blood making, blood forming) go back to a galaxy of eminent scientists and Nobel prizewinners of the early 20th century, such as Emil von Behring, Paul Ehrlich, Jules Bordet, Karl Landsteiner, Ilya Mechnikov... Thus, two German scientists, Ehrlich and von Behring, proved the existence of antibodies-the protective proteins synthesized by the organism in response to intruding foreign proteins. Jules Bordet of Belgium found that blood transfusion from one animal to another incompatible as to their biological species results in the hemagglutination (gluing together) of erythrocytes (red blood cells) and put it down to the action of antibodies. Paul Ehrlich observed a similar reaction within one and the same animal species. And Karl Landsteiner of Austria (one who discovered different groups of blood) demonstrated that red blood cell

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agglutination is a normal phenomenon, not a pathology. The Russian biologist and member of the St. Petersburg Academy of Sciences Ilya Mechnikov and coworkers were the first to obtain cytotoxic serums containing antibodies to red blood cells and leucocytes (white cells). His research team found that minor doses of hemolytic serum stimulate erythropoiesis (formation of red blood cells), and those of leucotoxin exert the same effect on leucopoiesis (formation of white blood cells). In the 1930s Acad. Alexander Bogomolets showed that the action of such serum is dose-dependent - a big dose inhibits the activity of tissues and organs and vice versa, a small one has a stimulating effect. So, on the one hand, the scientists proved the very possibility of antibodies being formed to organic cells and their high specificity (i.e. targeted, selective action); and on the other hand, they found that antibodies introduced from outside affect the functional activity of tissues (in this particular case, blood forming).

In 1932 Yakov Uzhansky, a young research worker employed by Dr. Bogomolets in his laboratory observed this phenomenon: that bleeding stimulated erythrophagocytosis in experimental rats and hens with the subsequent formation of a ferrous pigment. That is red blood cell making was stimulated thereby. This work was in fact the first study indicating the possible role of phagocyte (scavenger) cells in hemopoietic regulation.

Then, in 1947, Pyotr Grabar (a Russian researcher working in France) suggested that under normal conditions the organism should carry natural autoantibodies which perform both the immune and the physiological functions. These antibodies step up phagocytosis and help the organism get rid of dead cells. Old cells are the first target of such scavengers. What is the cause of this selectivity? Some authors attribute it to the presence of neuraminic (sialic) acid on the cell membrane surface. Blood cells (red cells in particular) lose this acid in the process of ageing and cannot protect themselves against antibodies.

Today we are well aware of the multiple effect of the immune system on hemopoiesis. Their close interconnection has been also confirmed by experiments with cytotoxic serums. For instance, antilymphocyte globulin abolishes the suppression of allogenic stem cells* by lymphocytes, while antilymphocyte serum impairs lymphocytes capable of interaction with stem cells; and

Now we have a body of data on the implication of most of the cells of the immune system in the regulation of hemopoiesis both under normal physiological conditions and when the organism is affected by adverse factors. For example, macrophages produce erythropoietins stimulating the formation of erythrocytes (red blood cells); together with lymphocytes, they constitute what we call the colony-stimulating factor (CSF); the spleen inhibits leucopoiesis (formation of white blood cells) and erythropoiesis (formation of red blood cells); erythropoiesis is also inhibited by the lymphatic system.

LYMPHOCYTES

These cells are always present in the blood-making organs. In the bone marrow they occur in T- and B-forms. The significance of lymphocytes in sustaining hemopoiesis under normal physiological conditions and especially in extreme situations has long been studied in the laboratories of Acad. Rem Petrov and Acad. Yevgeny Goldberg (Russian Academy of Medicine). Acad. Pyotr Gorizontov (Russian Academy of Medicine) has likewise made a great contribution in this research by his studies in the 1960s, 1970s and 1980s.

The first data on the dependence of the functional activity of stem cells on direct interaction with lymphocytes were obtained in 1967 when Acad. Rem Petrov found that the joint transplantation of suspensions containing lymph cells of two genotypes suppresses the colony-forming units (CFU) present in them. Acad. Rem Petrov and his coworkers also noticed a change in the direction of differentiation of stem blood-forming cells-predominantly from the erythrocytic to the myeloid one - during their interaction with lymphocytes under the effect of antigen stimulation. Such stem cells intensify their proliferation (growth) and change their directivity not only under the effect of a mixture of different lymph cell species (T - and B-forms and other elements of the spleen) but also under the action of a sufficiently pure population of T-cells (suspension of thymus gland lymphocytes). The latter migrate into the bone marrow where the number of CFU increases under stress (the very fact of such migration is well established now).

It would be interesting to compare hemopoiesis in mice with congenital pathologies: devoid of thymus (Nude), devoid of spleen (Asplenic), and devoid of both (Lasat). The Nude and Lasat show progressive lymphopenia (decreasing number of

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lymphocytes), while in the Asplenic the number of lymphocytes is much higher than in normal individuals. The amount of bone marrow CFU in the Nude and Asplenic mice does not differ much from that registered in normal animals, while with the Lasat mice it is much reduced per femur (thigh). Unlike the Asplenic and Lasat mice, the Nude show an increase of granulocyte growth cells. The Lasat mice show a pronounced suppression of bone marrow erythropoiesis (formation of red blood cells). Acad. Goldberg and his pupils demonstrated that the bone marrow of the Nude mice has a much lower (1.5 - 3 fold) number of lymphocytes and erythroid elements, while their spleen reveals a decrease in the number of blasts and small lymphocytes, and an increase in the number of granulocytes, megacaryocytes and reticulocytes.

The blood-making process is much affected if some of the lymphoid organs are removed. In the absence of the thymus gland, mice and rats lag in the maturation of bone marrow tissue, as shown by the morphologic increase in the number of undifferentiated blasts and immature erythroid cells. Acad. Gorizontov and coauthors have cited data on the impact of thym - and splenectomy (removal of the thymus and spleen) on hemopoiesis in pubescent Wistar rats: observed for 60 days, the animals subjected to thymectomy had the same number of cell populations (lymph cells too) in the bone marrow as those in the control group. There were no changes in the number of erythrocytes and neutrophilic leucocytes in peripheric blood. Yet the number of lymphocytes showed a decrease.

Thymectomy in pubescent mice slows down the migration of stem cells to the spleen from an irradiated section of the bone marrow. Thereby the spleen produces only erythroid colonies by and large.

Splenectomy (removal of the spleen) causes an increase in the number of cells in the femur bone marrow due to the growth in the amount of erythroid and granulocyte elements; however, the number of lymph cells remains level. The peripheric blood of such rats shows a higher presence of leucocytes on account of lymphocytes and neutrophiles, which phenomenon is explained by the compensatory hypertrophy of lymph nodes.

The lower presence of thymocytes in spleen-resected rats is viewed as an indirect indicator of intensified migration of cells from the thymus gland upon splenectomy.

Abundant data on the immediate effect of all these blood cells on hemopoietic elements argue well for

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the regulatory role of lymphocytes in blood formation. The influence of lymphocytes on the proliferation and differentiation of stem hemopoietic cells is now supported by complementary evidence. The regulatory effect of lymphocytes in hemopoiesis, as it turns out, also depends on the initial status of the bone marrow and on the functional competence of lymphocytes, too.

The testing of the effect of interaction of CFU isolated from the intact, or "quiescent", bone marrow with identical T-cells in a syngenetic system ( a mix of genetically identical cells) causes no increase in the number of colonies in recipient spleens. These thymocytes affect no colony formation either in the activated bone marrow obtained from animals with thermal injuries. In fact, the addition of thymic cells to a transplant (1.8 g of bone marrow cells exposed to radiation) doubles the number of splenic colonies accompanied by a simultaneous increase in the CFU potential.

We can well postulate the presence of both T-cell helpers and those inhibiting colony formation in the hemopoietic tissue.

The regulatory effect of lymphocytes in blood formation should largely depend on the action of inducing factors and their nature. Say, in case of hypoxic hypoxia the injection of cobalt-treated T-lymphocytes stimulates the proliferation and differentiation of bone marrow erythroid cells.

Thus we see a close connection between the protective and hemopoiesis-regulating functions of lymphocytes when the conventional immunological effects on the activity of different types of lymph cells cause change in their hemopoietic competence. But these are only a part of the processes responsible for the steady balance of blood cells in the organism.

MONOCYTES/MACROPHAGES

As shown by many experiments and clinical studies, these cells have an important role to play in hemopoiesis. Thus, the bone marrow of adult mammalians contains characteristic erythroblast islets, or anatomical units, with a macrophage in the center. The macrophage is surrounded by one or several layers of erythroid cells. Located in the center may also be a monocyte or its precursor, a monoblast. These structures differ in the degree of maturity. The bone marrow of healthy humans contains as many as 137 erythroblast islets per 1 mg of tissue. Their number drops severalfold if erythro-poiesis is suppressed, but it goes up should the formation of red blood cells be stimulated. The insular macrophages play a major part in the physiology of erythroid cells by acting upon their proliferation and growth. Granulocyte and erythroidgranulocyte islets have also been identified.

Monocyte/macrophage cells produce erythropoietin, prostaglandins, the tumor necrosis factor, inter-leukins and possibly, some other substances poorly identified so far but capable of stimulating and suppressing erythropoiesis alike. Monocytes/macrophages synthesize all types of CFU known to us. Such productivity increases with the maturation of monocytes, and it intensifies dramatically if activated by bacterial products and T-lymphocytes.

Since there is no doubt about the implication of monocytes/macro-phages in hemopoiesis, with phagocytosis being their key function in the immune defenses, it is important to find out how these two processes are interconnected. It has been proved that blood cell decomposition products are also involved in the physiological mechanisms of hemopoiesis. The destruction of blood cells is usually related to humoral and cell immunity. The bulk of decomposition products is the target of phagocytosis. This brings us to another question-how the dead blood cells are scavenged by a system of phagocyte mononuclears during blood regeneration.

NEUTROPHILES

Since this type of leucocytes has a wide range of means to act upon macrophages in a variety of ways, it would be in place to consider the idea of the hemopoietic implication of these blood cells. Their secretory products should exert a modulating influence on the functions of the central macrophage of blood-making islets of the bone marrow (erythroblasts in particular). Supernatant-activated neutrophiles, when administered to intact animals, have no effect on the amount of erythrocytes and hemoglobin but cause a dose-dependent increase in the concentration of reticulocytes (immature red blood cells).

Significant changes are observed in the bone marrow. Thus, 0.2 - 1.0 ml doses of supernatant of activated neutrophiles cause an absolute increase in the number of erythroblast islets. In animals having a normal level of erythropoiesis the same secretory products have a stimulating effect on it, speeding up the amplification (propagation) wave that brings a large number of young reticulocytes into the peripheric blood channel.

LABROCYTES, MASTOCYTES

Yet another cell type within the immune system is represented by mast cells-labrocytes and mastocytes. Under normal physiological conditions their location in the bone marrow is endosteal, i.e. within the marrow, though their concentration in the center of the bone marrow canal is not high.

A stereotypic reaction is observed if the organism is acted upon by extreme factors (acute and chronic hypoxia, normobaric hyperoxia, radiation) when the absolute number of labrocytes in the bone marrow diminishes. This may be viewed as a stress-conditioned reaction, for something like that occurs under classical immobilization stress; the effect is less pronounced in animals subjected to adrenalectomy (i.e. with the adrenal cortex excised). A drop in the total amount of labro-

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cytes in the femur (thighbone) is caused by hydrocortisone, adrenalin, histamine, and heparin; the cells disappear altogether under the action of acetylcholine.

Mast cells grow in diameter under any effects except inflammation when their size remains the same.

Histamine causes a contraction in the overall volume of bone marrow vessels. Their sensitivity to it is suggestive of the possible role of mast cells in the regulation of bone marrow hemodynamics. The same compound increases the permeability of vessels by acting upon the endothelial cells of postcapillary venules (- 20 - 30 μ in diameter) and may be involved in bringing blood cells into circulation. Vascular cells can also produce a group of hemopoiesis regulators assigned to glycoproteins.

Histamine has an explicit inhibitory effect on bone marrow fibroblasts by slowing down their growth rate in a culture medium and reducing their number in mitotic division (the effect is dose-dependent); besides, these cells no longer adhere readily to glass.

Histamine acts directly on hemopoietic cells. Thus, it activates the processes of the proliferation and differentiation of granulocyte precursors via H2-receptors (i.e. receptors to 2 type histamine). If bone marrow cells are cultivated for 3 days in the presence of histamine at a concentration of 10-5 - 10-8 mol, the growth of cell colonies from leucocyte precursors is stimulated. But the histamine effect is abolished with the simultaneous use of preparations blocking H2-receptors (like cymetidin or rhanitidin). Chemokinesis and chemotaxis are inhibited thereby, with lysosomal enzymes liberated and superoxydanion being formed by in vitro cultivated human neutrophiles. These blood cells may also have HI receptors (relative to 1 type histamine) which mediate the chemoattractive effect of histamine on human eosinophiles (stimulation of H2-receptors has the reverse effect); in addition, it activates the production of superoxydanion-radical O2 by human and guinea pig eosinophiles. HI-receptors have been found on monocytes and H2 ones - on basophiles (basophilic leu-

The role of mast cells in hemopoiesis.

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cocytes). In this case histamine has an inhibitory effect on the functions of neutrophiles, eosinophiles and basophiles.

Lymph cells (lymphocytes) apparently possess both HI - and H2-receptors depending on the animal species and lymphocyte population. H2-receptors are present on cytotoxic T-lymphocytes and natural killers, on lymphocyte and antibody producers, and on T-suppressors. Stimulation of lymphocyte H2-receptors inhibits the synthesis or release of immunoglobulins, lymphokins as well as cytotoxicity of T-cells.

Heparin has an analogous effect on a fibroblast culture. In this case, too, the effect is dosage-dependent, though the activity of histamine is lower.

Heparin sulfate, a representative of glycosamineglycanes, inhibits the sensitivity of CFU-E (colony-forming units of erythroids) to erythropoietin. Together with the stimulating factor, the presence of heparin sulfate is essential for the optimal growth of fibroblast colonies.

Finally, D-glucuronic acid is selective in stimulating processes of bone marrow granulocytopoiesis in intact animals. And in mice with cyclophosphane-suppressed hemopoiesis this acid also actuates formation of a significant number of new hemopoietic islets composed of a central macrophage or fibroblast and surrounded by blood-making elements. D-glucuronic acid assists in the formation of predominantly granulocyte islets, and acts upon the proliferation of hemopoietic cells through the system of prostaglandins.

EOSINOPHILES

Leucocytes of this type are assigned the role of the chief protective mechanism against larval forms of parasitosis. They are thought to act as modulators of hypersensitivity reactions (e.g. allergy). But the organism needs the system of eosinophiles for many other purposes as well. Only 10 - 25 percent of eosinophiles contain receptors to IgE (E class immunoglobulin, a variety of antibodies), though their number tends to increase in hypereosinophilia.

By now data have been obtained on the competence of eosinophiles as part of the immune regulatory system. They produce a complex of physiologically active substances impacting hemopoiesis. Eosinophiles can generate leucotrienes (oxidation products of arachodonic acid) as well as polysaccharide molecules-glycosamineglycanes (mucopolysaccharides), chondroitin-4-sulfate in particular; these substances can synthesize one of the enzymes implicated in blood clot destruction-profibrinolysine and the platelet-activating factor. Their involvement in blood coagulation and in hemostasia (arrest of bleeding) can hardly be great, though it may become essential in sustaining the aggregate condition in the microcirculatory blood channel, especially under normal conditions.

Eosinophiles and mast cells are supposed to form an integral system of hemopoietic regulation. Eosinophiles are largely responsible for the production of mast cells and their future life. They can phagocytose granules secreted by mast cells due to the presence of proteinases cleaving leucotrienes С and D. One of the possible functions of eosinophiles (possibly prostaglandin E2 -mediated) is the direct suppression of histamine secretion. Though implicated in histamine metabolism, they do not synthesize but only phagocytose and accumulate granules produced by mast cells and basophiles. The ability of eosinophiles to adsorb histamine on their surface is disputed though. Yet this possibility holds on account of the presence of histamine-specific receptors on their membranes.

The correlation of labrocytes, mastocytes and eosinophiles is regulated both by external mechanisms and by intercellular interaction. For instance, the number of mast cells decreases in the bone marrow of adrenalectomized animals, while that of eosinophiles is up.

In turn, the hemopoiesis affects the immune system as well. This feedback is observed as lymph cells, contacting syngenetic erythrocytes, secrete immunosuppression-competent factors; bloodletting stimulates formation of antibodies but suppresses their activity; stimulation or suppression of erythropoiesis affects both the genesis of antibodies and the migration of B-cells from the bone marrow to the spleen. Which means that erythropoietin can act as a regulator of antibody genesis.

In the 1970s biologists of this country isolated substances from the bone marrow-myelopeptides* - impacting the functional competence of stem hemopoietic cells and the activity of the immunity B-system which stimulates antibody formation at the peak of an immune response. Myelopeptides also act upon the cytolytic activity of T-cell. They are more productive under stress, in particular, during immobilization (due to excitation of the hemopoietic system). The interaction of both systems is predicated on the common origin of their cells organelles from a single stem blood cell.

Thus, the immune system is playing a major role in the regulation of hemopoiesis. Let us stress that the control over its processes is exercised by means of immunological reactions and other mechanisms.